Reduced Levels of H2S in Diabetes-Associated Osteoarthritis Are Linked to Hyperglycaemia, Nrf-2/HO-1 Signalling Downregulation and Chondrocyte Dysfunction.

Different findings indicate that type 2 diabetes is an independent risk factor for osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Changes in the balance of hydrogen sulphide (H2S) are thought to play an important role in the pathogenesis of diabetes and its complications, although its role is still controversial. In this study, we examined the modulation of H2S levels in serum and chondrocytes from OA diabetic (DB) and non-diabetic (non-DB) patients and in cells under glucose stress, in order to elucidate whether impairment in H2S-mediated signalling could participate in the onset of DB-related OA. Here, we identified a reduction in H2S synthesis in the cartilage from OA-DB patients and in cells under glucose stress, which is associated with hyperglycaemia-mediated dysregulation of chondrocyte metabolism. In addition, our results indicate that H2S is an inductor of the Nrf-2/HO-1 signalling pathway in cartilage, but is also a downstream target of Nrf-2 transcriptional activity. Thereby, impairment of the H2S/Nrf-2 axis under glucose stress or DB triggers chondrocyte catabolic responses, favouring the disruption of cartilage homeostasis that characterizes OA pathology. Finally, our findings highlight the benefits of the use of exogeneous sources of H2S in the treatment of DB-OA patients, and warrant future clinical studies.

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy.

BACKGROUND: Ageing-related failure of homeostasis mechanisms contributes to articular cartilage degeneration and osteoarthritis (OA), for which disease-modifying treatments are not available. Our objective was to identify molecules to prevent OA by regulating chondrocyte senescence and autophagy. METHODS: Human chondrocytes with IL-6 induced senescence and autophagy suppression and SA-ß-gal as a reporter of senescence and LC3 as reporter of autophagic flux were used to screen the Prestwick Chemical Library of approved drugs. Preclinical cellular, tissue and blood from OA and blood from OA and ageing models were used to test the efficacy and relevance of activating PPARα related to cartilage degeneration. FINDINGS: Senotherapeutic molecules with pro-autophagic activity were identified. Fenofibrate (FN), a PPARα agonist used for dyslipidaemias in humans, reduced the number of senescent cells via apoptosis, increased autophagic flux, and protected against cartilage degradation. FN reduced both senescence and inflammation and increased autophagy in both ageing human and OA chondrocytes whereas PPARα knockdown conferred the opposite effect. Moreover, PPARα expression was reduced through both ageing and OA in mice and also in blood and cartilage from knees of OA patients. Remarkably, in a retrospective study, fibrate treatment improved OA clinical conditions in human patients from the Osteoarthritis Initiative (OAI) Cohort. INTERPRETATION: These results demonstrate that FDA-approved fibrate drugs targeting lipid metabolism protect against cartilage degeneration seen with ageing and OA. Thus, these drugs could have immediate clinically utility for age-related cartilage degeneration and OA treatment. FUND: This study was supported by Instituto de Salud Carlos III- Ministerio de Ciencia, Innovación y Universidades, Spain, Plan Estatal 2013-2016 and Fondo Europeo de Desarrollo Regional (FEDER), "Una manera de hacer Europa", PI14/01324 and PI17/02059, by Innopharma Pharmacogenomics platform applied to the validation of targets and discovery of drugs candidates to preclinical phases, Ministerio de Economía y Competitividad, by grants of the National Instiutes of Health to PDR (P01 AG043376 and U19 AG056278). We thank FOREUM Foundation for Research in Rheumatology for their support.

Role of the Inflammation-Autophagy-Senescence Integrative Network in Osteoarthritis.

Osteoarthritis is the most common musculoskeletal disease causing chronic disability in adults. Studying cartilage aging, chondrocyte senescence, inflammation, and autophagy mechanisms have identified promising targets and pathways with clinical translatability potential. In this review, we highlight the most recent mechanistic and therapeutic preclinical models of aging with particular relevance in the context of articular cartilage and OA. Evidence supporting the role of metabolism, nuclear receptors and transcription factors, cell senescence, and circadian rhythms in the development of musculoskeletal system degeneration assure further translational efforts. This information might be useful not only to propose hypothesis and advanced models to study the molecular mechanisms underlying joint degeneration, but also to translate our knowledge into novel disease-modifying therapies for OA.

The relationship of autophagy defects to cartilage damage during joint aging in a mouse model.

OBJECTIVE: Aging is a main risk factor for osteo arthritis (OA), the most prevalent musculoskeletal disorder. Defects in autophagy, an essential cellular homeostasis mechanism, have recently been observed in OA articular cartilage. The objectives of this study were to establish the constitutive level of autophagy activation in normal cartilage and to monitor the temporal relationship between changes in autophagy and aging-related degradation of cartilage in a mouse model. METHODS: In GFP-LC3-transgenic mice, green fluorescent protein (GFP)-light chain 3 (LC3) is ubiquitously expressed, and the accumulation of GFP puncta, representing autophagosomes, was quantified by confocal microscopy as a measure of autophagy activation. Expression of the autophagy proteins autophagy-related protein 5 (ATG-5) and microtubule-associated protein 1 light chain 3 (LC3) was analyzed by immunohistochemistry. Cartilage cellularity, apoptotic cell death, and cartilage structural damage and changes in synovium and bone were examined by histology and immunohistochemistry. RESULTS: Basal autophagy activation was detected in liver and knee articular cartilage from young (6-month-old) mice, with higher levels in cartilage than in liver in the same animals. In 28-month-old mice, there was a statistically significant reduction in the total number of autophagic vesicles per cell (P < 0.01) and in the total area of vesicles per cell (P < 0.01) in the articular cartilage as compared to that from young 6-month-old mice. With increasing age, the expression of ATG-5 and LC3 decreased, and this was followed by a reduction in cartilage cellularity and an increase in the apoptosis marker poly(ADP-ribose) polymerase p85. Cartilage structural damage progressed in an age-dependent manner subsequent to the autophagy changes. CONCLUSION: Autophagy is constitutively activated in normal cartilage. This is compromised with aging and precedes cartilage cell death and structural damage.

Autophagy activation and protection from mitochondrial dysfunction in human chondrocytes.

OBJECTIVE: Autophagy is a key pathway of cellular homeostasis for removing damaged macromolecules and organelles, including mitochondria. Recent studies indicate that activation of autophagy is defective in aging and osteoarthritis (OA), contributing to cell death and tissue damage. In addition, there is increasing evidence that mitochondrial dysfunction plays an important role in OA pathogenesis. The objective of this study was to determine whether activation of autophagy protects against mitochondrial dysfunction in human chondrocytes. METHODS: Human chondrocytes were treated with oligomycin, an inhibitor of mitochondrial respiratory chain complex V. Autophagy activation was analyzed by determination of light chain 3 membrane-bound form II (LC3-II), a marker of autophagosome formation. To investigate whether autophagy protects from mitochondrial dysfunction, autophagy was induced by rapamycin, the selective inhibitor of mammalian target of rapamycin complex 1 (mTORC-1), and by torin 1, the inhibitor of mTORC-1 and mTORC-2. Small interfering autophagy-related 5 was used to evaluate the role of autophagy in mitochondrial dysfunction. RESULTS: Mitochondrial dysfunction was induced by treatment with oligomycin, which significantly decreased mitochondrial membrane potential (ΔΨm). This was associated with increased production of reactive oxygen species and cell death. Autophagy activation, as reflected by LC3-II, was decreased in a time-dependent manner. To evaluate whether autophagy regulates mitochondrial function, chondrocytes were pretreated with rapamycin and torin 1 before oligomycin. Autophagy activation significantly protected against mitochondrial dysfunction. Conversely, genetic inhibition of autophagy induced significant mitochondrial function defects. CONCLUSION: Our data highlight the role of autophagy as a critical protective mechanism against mitochondrial dysfunction. Pharmacologic interventions that enhance autophagy may have chondroprotective activity in cartilage degenerative processes such as OA.

FoxO transcription factors support oxidative stress resistance in human chondrocytes.

OBJECTIVE: A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes. METHODS: Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1ß were assessed. Cells transfected with FOXO plasmids were also analyzed. RESULTS: Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes. CONCLUSION: Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

Glucosamine activates autophagy in vitro and in vivo.

OBJECTIVE: Aging-associated changes in articular cartilage represent a main risk factor for osteoarthritis (OA). Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimentally induced defects in autophagy contribute to organismal- and tissue-specific aging, while enhancement of autophagy may protect against certain aging-related pathologies such as OA. The objective of this study was to determine whether glucosamine can activate autophagy. METHODS: Chondrocytes from normal human articular cartilage were treated with glucosamine (0.1- 10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3, and ribosomal protein S6 were determined by Western blotting. Autophagosome formation was analyzed by confocal microscopy. Reporter mice systemically expressing green fluorescent protein (GFP) fused to light chain 3 (LC3) (GFP-LC3-transgenic mice) were used to assess changes in autophagy in response to starvation and glucosamine treatment. RESULTS: Glucosamine treatment of chondrocytes activated autophagy, as indicated by increased LC3-II levels, formation of LC3 puncta, and increased LC3 turnover. This was associated with glucosamine-mediated inhibition of the Akt/FoxO3/mammalian target of rapamycin pathway. Administration of glucosamine to GFP-LC3-transgenic mice markedly activated autophagy in articular cartilage. CONCLUSION: Glucosamine modulates molecular targets of the autophagy pathway in vitro and in vivo, and the enhancement of autophagy is mainly dependent on the Akt/FoxO/mTOR pathway. These findings suggest that glucosamine is an effective autophagy activator and should motivate future studies on the efficacy of glucosamine in modifying aging-related cellular changes and supporting joint health.

Autophagy activation by rapamycin reduces severity of experimental osteoarthritis.

OBJECTIVES: Osteoarthritis is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in ageing and osteoarthritic cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental osteoarthritis. METHODS: Experimental osteoarthritis was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month-old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or dimethyl sulphoxide vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signalling, autophagy, cartilage homeostasis and inflammation were analysed by immunohistochemistry and immunofluorescence staining. RESULTS: Rapamycin affected the mTOR signalling pathway in mouse knee joints as indicated by the inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (p<0.01) reduced in the rapamycin-treated group compared with the control group and this was associated with a significant (p<0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and interleukin-1ß expression in articular cartilage. CONCLUSIONS: These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental osteoarthritis. Pharmacological activation of autophagy may be an effective therapeutic approach for osteoarthritis.

Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection.

OBJECTIVE: Mechanical injury induces cell death in cartilage and triggers a remodeling process that ultimately can manifest as osteoarthritis. Autophagy is a process for turnover of intracellular organelles and macromolecules that protects cells during stress responses. This study was undertaken to determine changes in and functions of autophagy following mechanical injury to cartilage. METHODS: Bovine and human cartilage explants were subjected to mechanical impact (40% strain for 500 msec). Cell viability, sulfated glycosaminoglycan (sGAG) release, and changes in the levels of the autophagy markers ULK1, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3) were evaluated. Cartilage explants were treated with the mammalian target of rapamycin complex 1 (mTORC-1) inhibitor and the autophagy inducer rapamycin and tested for protective effects against mechanical injury. Explants were also treated with the cell death inducers nitric oxide and tumor necrosis factor α (TNFα) plus actinomycin D, and the proinflammatory cytokine interleukin-1α (IL-1α). RESULTS: Mechanical injury induced cell death and loss of sGAG in a time-dependent manner. This was associated with significantly decreased ULK1, beclin 1, and LC3 expression in the cartilage superficial zone (P < 0.05) 48 hours after injury. The levels of LC3-II were increased 24 hours after injury but decreased at 48 and 96 hours. Rapamycin enhanced expression of autophagy regulators and prevented cell death and sGAG loss in mechanically injured explants. Rapamycin also protected against cell death induced by sodium nitroprusside and TNFα plus actinomycin D and prevented sGAG loss induced by IL-1α. CONCLUSION: Our findings indicate that mechanical injury leads to suppression of autophagy, predominantly in the superficial zone where most of the cell death occurs. Pharmacologic inhibition of mTORC-1, at least in part by enhancement of autophagy, prevents cell and matrix damage, suggesting a novel approach for chondroprotection.

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation.

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/ß-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.

Autophagy and cartilage homeostasis mechanisms in joint health, aging and OA.

Osteoarthritis (OA) is the most prevalent joint disease, but neither preventive measures nor disease-modifying drugs are available and a continuing need exists for safe and effective symptom-modifying therapies. Clinical trials of candidate disease-modifying OA drugs in patients with established or advanced disease have not demonstrated their efficacy, but these failed trials have motivated investigation into the mechanisms that maintain joint health. The enhancement of such mechanisms could be a novel approach to reducing the risk of OA. Aging is one of the most important risk factors for OA; however, aging of joint cartilage is a process that is distinct from the subsequent cartilage changes that develop following the onset of OA. This Review focuses on the mechanisms that maintain cell and tissue homeostasis, and how these mechanisms fail during the aging process. Autophagy is a cellular homeostasis mechanism for the removal of dysfunctional organelles and macromolecules. Defective autophagy is involved in the pathogenesis of aging-related diseases and recent observations indicate that this process is compromised in aging cartilage. Augmentation of homeostasis mechanisms is discussed as a novel avenue to delay joint aging and reduce OA risk.

Proteomic analysis by two-dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor alpha and interleukin-1beta.

OBJECTIVE: To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) and to ascertain differences in the protein expression patterns of these 2 cytokines. METHODS: Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL-1beta (5 ng/ml) or with TNFalpha (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2-dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology. RESULTS: We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL-1beta-stimulated chondrocytes, IL-1beta was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up-regulated. Proteins involved in cellular metabolism and energy (23%) that were up-regulated or induced by IL-1beta included nicotinamide phosphoribosyltransferase, long-chain fatty acid-coenzyme A ligase 4, delta-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNFalpha-stimulated chondrocytes, TNFalpha was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3-like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up-regulated. Eighteen proteins were differently modulated by IL-1beta and TNFalpha. Of these, 45% were related to metabolism. CONCLUSION: IL-1beta and TNFalpha induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis.

OBJECTIVE: Autophagy is a process for turnover of intracellular organelles and molecules that protects cells during stress responses. We undertook this study to evaluate the potential roles of Unc-51-like kinase 1 (ULK1), an inducer of autophagy, Beclin1, a regulator of autophagy, and microtubule-associated protein 1 light chain 3 (LC3), which executes autophagy, in the development of osteoarthritis (OA) and in cartilage cell death. METHODS: Expression of ULK1, Beclin1, and LC3 was analyzed in normal and OA human articular cartilage and in knee joints of mice with aging-related and surgically induced OA, using immunohistochemistry and Western blotting. Poly(ADP-ribose) polymerase (PARP) p85 expression was used to determine the correlation between cell death and autophagy. RESULTS: ULK1, Beclin1, and LC3 were constitutively expressed in normal human articular cartilage. ULK1, Beclin1, and LC3 protein expression was reduced in OA chondrocytes and cartilage, but these 3 proteins were strongly expressed in the OA cell clusters. In mouse knee joints, loss of glycosaminoglycans (GAGs) was observed at ages 9 months and 12 months and in the surgical OA model, 8 weeks after knee destabilization. Expression of ULK1, Beclin1, and LC3 decreased together with GAG loss, while PARP p85 expression was increased. CONCLUSION: Autophagy may be a protective or homeostatic mechanism in normal cartilage. In contrast, human OA and aging-related and surgically induced OA in mice are associated with a reduction and loss of ULK1, Beclin1, and LC3 expression and a related increase in apoptosis. These results suggest that compromised autophagy represents a novel mechanism in the development of OA.

Chromatin protein HMGB2 regulates articular cartilage surface maintenance via beta-catenin pathway.

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis. Mechanisms that regulate the unique phenotype of SZ chondrocytes and maintain SZ integrity are unknown. We recently demonstrated that expression of the chromatin protein high mobility group box (HMGB) protein 2 is restricted to the SZ in articular cartilage suggesting a transcriptional regulation involving HMGB2 in SZ. Here, we show that an interaction between HMGB2 and the Wnt/beta-catenin pathway regulates the maintenance of the SZ. We found that the Wnt/beta-catenin pathway is active specifically in the SZ in normal mouse knee joints and colocalizes with HMGB2. Both Wnt signaling and HMGB2 expression decrease with aging in mouse joints. Our molecular studies show that HMGB2 enhances the binding of Lef-1 to its target sequence and potentiates transcriptional activation of the Lef-1-beta-catenin complex. The HMG domain within HMGB2 is crucial for interaction with Lef-1, suggesting that both HMGB2 and HMGB1 may be involved in this function. Furthermore, conditional deletion of beta-catenin in cultured mouse chondrocytes induced apoptosis. These findings define a pathway where protein interactions of HMGB2 and Lef-1 enhance Wnt signaling and promote SZ chondrocyte survival. Loss of the HMGB2-Wnt signaling interaction is a new mechanism in aging-related cartilage pathology.

Aging-related loss of the chromatin protein HMGB2 in articular cartilage is linked to reduced cellularity and osteoarthritis.

Osteoarthritis (OA) is the most common joint disease and typically begins with an aging-related disruption of the articular cartilage surface. Mechanisms leading to the aging-related cartilage surface degeneration remain to be determined. Here, we demonstrate that nonhistone chromatin protein high-mobility group box (HMGB) protein 2 is uniquely expressed in the superficial zone (SZ) of human articular cartilage. In human and murine cartilage, there is an aging-related loss of HMGB2 expression, ultimately leading to its complete absence. Mice genetically deficient in HMGB2 (Hmgb2(-/-)) show earlier onset of and more severe OA. This is associated with a profound reduction in cartilage cellularity attributable to increased cell death. These cellular changes precede glycosaminoglycan depletion and progressive cartilage erosions. Chondrocytes from Hmgb2(-/-) mice are more susceptible to apoptosis induction in vitro. In conclusion, HMGB2 is a transcriptional regulator specifically expressed in the SZ of human articular cartilage and supports chondrocyte survival. Aging is associated with a loss of HMGB2 expression and reduced cellularity, and this contributes to the development of OA.

Mitochondrial dysfunction activates cyclooxygenase 2 expression in cultured normal human chondrocytes.

OBJECTIVE: Mitochondrial alterations play a key role in the pathogenesis of osteoarthritis (OA). This study evaluated a potential role of mitochondrial respiratory chain (MRC) dysfunction in the inflammatory response of normal human chondrocytes. METHODS: Commonly used inhibitors of the MRC were utilized to induce mitochondrial dysfunction in normal human chondrocytes. Levels of prostaglandin E(2) (PGE(2)) protein and expression of cyclooxygenase 2 (COX-2) and COX-1 messenger RNA (mRNA) and protein were analyzed. To identify the underlying mechanisms responsible for PGE(2) liberation, reactive oxygen species (ROS) were measured. Inhibitors of ROS, including vitamin E, and inhibitors of mitochondrial Ca(2+) and NF-kappaB were used to test their effects on the MRC. RESULTS: Antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) significantly increased the levels of PGE(2) (mean +/- SEM 505 +/- 132 pg/50,000 cells and 288 +/- 104 pg/50,000 cells, respectively, at 24 hours versus a basal level of 29 +/- 9 pg/50,000 cells; P < 0.05) and increased the expression of COX-2 at both the mRNA and protein levels. Expression of COX-1 did not show any modulation with either inhibitor. Further experiments revealed that antimycin A and oligomycin induced a marked increase in the levels of ROS. Production of PGE(2) and expression of COX-2 protein were inhibited by antioxidants, vitamin E, and mitochondrial Ca(2+) and NF-kappaB inhibitors. The response to blockers of mitochondrial Ca(2+) movement showed that ROS production was dependent on mitochondrial Ca(2+) accumulation. CONCLUSION: These results strongly suggest that, in human chondrocytes, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to PGE(2) production via mitochondrial Ca(2+) exchange, ROS production, and NF-kappaB activation. These data may prove valuable for a better understanding of the participation of mitochondria in the pathogenesis of OA.

Evidencia de mecanismos inflamatorios en la osteoartritis / Evidence of inflammatory mechanisms in osteoarthritis

Clásicamente, la artrosis (OA) no ha sido considerada una artropatía inflamatoria por la escasez de neutrófilos en el líquido sinovial y la ausencia de manifestaciones sistémicas de inflamación. Además, las características del cartílago articular (avascular, alinfático y aneural) impiden cumplir los signos clásicos de la inflamación (enrojecimiento, hinchazón, calor y dolor). Sin embargo, gracias a los avances en biología molecular y celular, son múltiples los estudios que demuestran que diversos mediadores proinflamatorios, como las citocinas interleucina 1 y factor de necrosis tumoral , pueden ser importantes en el desarrollo de esta enfermedad. Así, la estimulación de condrocitos, único representante del cartílago articular y por ello principal encargado de mantener la matriz extracelular del cartílago, con estas citocinas proinflamatorias incrementa la producción de las metaloproteasas, enzimas proteolíticas clave en la degradación irreversible de la arquitectura articular normal. También inhiben la síntesis de proteoglucanos y colágeno tipo II, estimulan la producción de especies reactivas de oxígeno como el óxido nítrico e incrementan la producción de prostaglandina E2. Asimismo, es evidente que los efectos de la inflamación sinovial favorecen la desregulación en la función del condrocito y la pérdida del equilibrio entre las actividades anabólicas y catabólicas del condrocito, imprescindibles para mantener la integridad articular normal (AU)

Classically, osteoarthritis (OA) is not considered an inflammatory arthropathy, because of the presence of a small number of neutrophils in the synovial fluid and the absence of systemic manifestations of inflammation. Besides, the characteristics of articular cartilage (avascular, alymphatic and aneural) do disable to fulfill with the classical signs of inflammation (redness, swelling, heat, pain). However, thanks to development of molecular and cellular biology, there are multiple studies which shown that different proinflammatory mediators, such as the cytokines IL-1 and TNF, could be important in the development of this disease. Therefore, the stimulation of chondrocytes, the only cell type living in the cartilage matrix and for this reason the principal responsible of integrity of cartilage matrix extracellular, with these proinflamatory cytokines increases the production of metalloproteinases, keys molecules in the irreversible degradation of normal architecture of cartilage. As well, inhibits the synthesis of cartilage proteoglycans and type II collagen, stimulates the production of reactive oxygen species such as nitric oxide, and increases the production of prostaglandin E2. Likewise, the effects of synovial inflammation expected contribute to deregulation of chondrocyte function in a similar fashion, favouring the lost of equilibrium between the catabolic and anabolic activities of the chondrocyte necessary for maintaining the extracellular cartilage matrix (AU)

[Evidence of inflammatory mechanisms in osteoarthritis]. / Evidencia de mecanismos inflamatorios en la osteoartritis.

Classically, osteoarthritis (OA) is not considered an inflammatory arthropathy, because of the presence of a small number of neutrophils in the synovial fluid and the absence of systemic manifestations of inflammation. Besides, the characteristics of articular cartilage (avascular, alymphatic and aneural) do disable to fulfill with the classical signs of inflammation (redness, swelling, heat, pain). However, thanks to development of molecular and cellular biology, there are multiple studies which shown that different proinflammatory mediators, such as the cytokines IL-1ß and TNFα, could be important in the development of this disease. Therefore, the stimulation of chondrocytes, the only cell type living in the cartilage matrix and for this reason the principal responsible of integrity of cartilage matrix extracellular, with these proinflamatory cytokines increases the production of metalloproteinases, keys molecules in the irreversible degradation of normal architecture of cartilage. As well, inhibits the synthesis of cartilage proteoglycans and type II collagen, stimulates the production of reactive oxygen species such as nitric oxide, and increases the production of prostaglandin E(2). Likewise, the effects of synovial inflammation expected contribute to deregulation of chondrocyte function in a similar fashion, favouring the lost of equilibrium between the catabolic and anabolic activities of the chondrocyte necessary for maintaining the extracellular cartilage matrix.